PROJECT SUMMARY: We are interested in understanding how lipid-metabolizing enzymes function and are regulated at the molecular and structural level. Our current focus is on two enzymes in phosphatidic acid (PA) metabolism: Lipin and phospholipase D (PLD). Lipins are lipid phosphatases that dephosphorylate PA to generate diacylglycerol, which is the penultimate step in triglyceride biosynthesis. Lipins regulate triglyceride biosynthesis, triglyceride catabolism, fatty acid synthesis, and insulin sensitivity with implications to obesity, diabetes, and cardiovascular disease. PLDs hydrolyze phosphatidylcholine to produce the lipid second messenger PA in response to extracellular stimuli. Receptor-mediated activation of PLD regulates vesicular trafficking, cell proliferation, and cell migration, which has established them as therapeutic targets for cancer. Both Lipin and PLD are highly regulated, multi-domain proteins that are structurally uncharacterized. Determining the three-dimensional structures of these enzymes alone, and in complex with their lipid substrates, lipid activators, and protein activators is essential to understanding their function and regulation. In preliminary data, we have used innovative bioinformatics and protein engineering to identify endogenous homologs and constructs of mouse/human Lipin and PLD that are more amenable for structural studies, demonstrated these constructs are fully functional in cells and in vitro, and obtained diffraction quality crystals. Structural studies will be complemented by an array of lipid biochemistry and lipid-protein interaction assays that we are well versed in, hydrogen-deuterium exchange mass spectrometry, and cellular studies in mammalian cells and yeast. A network of collaborators who are leaders in their respective fields supports these studies. We aim to answer several major questions: (1) How do lipid-modifying enzymes recognize their hydrophobic substrates and interact with the membrane during interfacial catalysis? (2) What role do novel structurally and functionally uncharacterized domains play in the action of these enzymes? (3) How do lipids activate these enzymes? (4) How are these enzymes regulated? Specifically, are they autoinhibited? How do protein effectors activate them? And what conformational changes occur during activation? Overall, this work will improve our understanding of biological mechanisms and provide information on lipid-protein interactions of physiological and pharmacological significance. In addition, this work will aid our long-term interest to develop and improve small molecule modulators of these enzymes, which would have potential therapeutic applications for the treatment of cancer, cardiovascular disease, and diabetes.